Amacrine cells are the most diverse group of retinal neurons. Various subtypes of amacrine interneurons mediate a vast majority of image forming and non-image forming visual functions. The transcriptional regulation governing the development of individual amacrine cell subtypes is not well understood. One such amacrine cell subtype comprises neuronal nitric oxide synthase (nNOS/bNOS/NOS1)-expressing amacrine cells (NOACs) that regulate the release of nitric oxide (NO), a neurotransmitter with physiological and clinical implications in the retina. We have identified the LIM-homeodomain transcription factor LHX9 to be necessary for the genesis of NOACs. During retinal development, NOACs express Lhx9, and Lhx9-null retinas lack NOACs. Lhx9-null retinas also display aberrations in dendritic stratification at the inner plexiform layer. Our cell lineage-tracing studies show that Lhx9-expressing cells give rise to both the GAD65 and GAD67 expressing sub-populations of GABAergic amacrine cells. As development proceeds, Lhx9 is downregulated in the GAD65 sub-population of GABAergic cells and is largely restricted to the GAD67 sub-population of amacrine cells that NOACs are a part of. Taken together, we have uncovered Lhx9 as a new molecular marker that defines a subset of amacrine cells and show that it is necessary for the development of the NOAC subtype of amacrine cells.

Retinal bipolar cells (BCs) connect with photoreceptors and relay visual information to retinal ganglion cells (RGCs). Retina-specific deletion of Lhx4 in mice results in a visual defect resembling human congenital stationary night blindness. This visual dysfunction results from the absence of rod bipolar cells (RBCs) and the loss of selective rod-connecting cone bipolar cell (CBC) subtypes and AII amacrine cells (ACs). Inactivation of Lhx4 causes the apoptosis of BCs and cell fate switch from some BCs to ACs, whereas Lhx4 overexpression promotes BC genesis. Moreover, Lhx4 positively regulates Lhx3 expression to drive the fate choice of type 2 BCs over the GABAergic ACs. Lhx4 inactivation ablates Bhlhe23 expression, whereas overexpression of Bhlhe23 partially rescues RBC development in the absence of Lhx4. Thus, by acting upstream of Bhlhe23, Prdm8, Fezf2, Lhx3, and other BC genes, Lhx4, together with Isl1, could play essential roles in regulating the subtype-specific development of RBCs and CBCs.

Haplo-insufficiency of the GATA3 gene causes hypoparathyroidism, sensorineural hearing loss, and renal disease (HDR) syndrome. Previous studies have shown that Gata3 is required for the development of the prosensory domain and spiral ganglion neurons (SGNs) of the mouse cochlea during embryogenesis. However, its role in supporting cells (SCs) after cell fate specification is largely unknown. In this study, we used tamoxifen-inducible Sox2CreERT2 mice to delete Gata3 in SCs of the neonatal mouse cochlea and showed that loss of Gata3 resulted in the proliferation of SCs, including the inner pillar cells (IPCs), inner border cells (IBCs), and lateral greater epithelium ridge (GER). In addition, loss of Gata3 resulted in the down-regulation of p27kip1, a cell cycle inhibitor, in the SCs of Gata3-CKO neonatal cochleae. Chromatin immunoprecipitation analysis revealed that GATA3 directly binds to p27kip1 promoter and could maintain the quiescent state of cochlear SCs by regulating p27kip1 expression. Furthermore, RNA-seq analysis revealed that loss of Gata3 function resulted in the change in the expression of genes essential for the development and function of cochlear SCs, including Tectb, Cyp26b1, Slitrk6, Ano1, and Aqp4.